Background: Children septicemia is a common cause of death in intensive care units in developing countries. However, determination of caused disease by blood culture was not effectively because using antibiotic of patients before they admitted hospital. Aims: apply multiplex real time PCR to diagnosis pathogen which caused septicemia id children in National Hospital of Peadiatric. Method: Analysis 30 children who from 1 month to 15 year old admitted Intensive care unit, NHP. Blood culture and multiplex real time PCR were using during from Jan to June, 2012. Result: Using multiplex real time PCR detected 43.3 percent positive patients but 3.3 percent detected by blood culture. The most common causal agent of children sepsis was